Tyrosine kinase.
It has 2 alpha and 2 beta subunits. The alpha subunits inhibit the beta subunits from phosphorylating. When insulin binds, the alpha subunits are changed so their inhibition is removed.

Name the 4 targets that the insulin receptor phosphorylates? Which 2 are the main ones?

IRS protein - main
CBL/Cap protein
Shc protein - main
Gab protein

What do the Sh2 proteins do?

The Sh2 proteins bind to phosphorylated tyrosine residues on the IRS protein. They are either kinases, phosphatases or activate G-protein cascades.

What are the roles of IRS1-IRS4?

IRS-1 is involved in cell differentiation and division due to insulin stimulus.
IRS-2 is involved in glucose uptake by the cell.
3 and 4 are unknown.

What is different about the way that the Cbl/Cap protein sends it's message?

It does not work through phosphorylation dependant kinase mechanisms. It activates lipid rafts and caveolae which increase glucose uptake in response to insulin.

What is PI3K? WHy is it called that? What happens when it does it's job?

PI3K is phosphatidylinositol 3 kinase and it is called that because it phosphorylates PIP2 at the 3' OH position.
When PIP2 is phosphorylated, it activates phosphorylation dependant kinases (PDK1). PDK1 increases glycogen synthesis, movement of GLUT4 to surface and cell division.

When it is activated by PI3K, it can activate phospholipase C which cleaves PIP2 into DAG and IP3. Both of these are 2nd messengers.

What mechanisms do the following proteins work through? IRS, Cbl/CAP, Shc and Gab.
What cellular changes can these make?

IRS works through PI3K to increase either PDK activity or phospholipase C activity. This stimulates glucose transport, glycogen synthesis and cell division.
Cbl/CAP works through lipid rafts/caveolae to increase glucose uptake.
Shc and Gab work through Mitogen activated protein kinases (MAPK) which stimulates cell division.

What are the 3 ways that IRS proteins are controlled?

Proteosome degradation
Dephosphorylation
Ser/thr phosphorylation

How does TNFa and ffa's cause insulin resistance in obese patients?

TNFa and ffa's stimulate ser/thr kinases which phosphorylate the IRS protein preventing it from being phosphorylated at tyr residues.
THIS DECREASES INSULIN SIGNAL

What is mTOR and how does it work?

mTOR is a protein product of the IRS PI3K cascade. It phosphorylates ser residues on the IRS protein which specifically prevents PI3K from binding to IRS.
This dampens the insulin signal through feedback inhibition.

How does PKCξ work?

It phosphorylates the IRS protein which causes it to release from the insulin receptor. IkB kinase β is the instigator that starts the whole process.

How do the protein tyrosine phosphatases relate to insulin sensitivity?

The PTPases are coded for by PTPB1. They dephosphorylate the IRS protein to decrease insulin signaling. KO mice for PTPB1 show increased insulin sensitivity.
An inhibitor PTPB1 is a potential drug therapy for obesity induced Type II.

How does the SHIP2 protein work?

It does exactly the opposite of PI3K, it dephosphorylates PIP2 at the 3' position.

What are potential treatments for insulin resistance?

Inhibitors of mTOR, PKCξ and IKKβ.
Salicylates have been shown to block IKKβ for treatment of type II.

What is the first step in ketone body formation? What enzyme is involved?

Add acetyl-CoA to acetoacetyl-CoA (the last 4 carbons of β oxidation) to form HMG-CoA.
HMG-CoA synthase

In ketone body generation instead of adding acetyl-CoA to acetoacetyl-CoA to form HMG-CoA why not just leave a 6 carbon fragment from β oxidation?

Because the acetyl-CoA is not added in a straight chain. It is added to the C=O on the β carbon where a OH group has now formed.

What do we do with HMG-CoA when we are making ketones?

Using HMG-CoA lyase, we remove acetyl-CoA leaving us with acetoacetate.

T/F the acetyl-CoA added to form HMG-CoA is the same acetyl-CoA that gets kicked off when formed ketone bodies?

False, it is a different 2C fragment.

What enzyme and cofactor are required for formation of β-hydroxybutyrate from acetoacetate?

β-hydroxybutyrate dehydrogenase and NADH is oxidized to NAD.

What is the difference in structure between β-hydroxybutyrate and acetoacetate?

β-hydroxybutyrate is a straight chain whereas acetoacetate is branched. The β carbon also has a hydroxyl instead of C=O which is why we need NADH.

How do we form acetone?

It is spontaneous decarboxylation from acetoacetate

What happens to ketones once they get to other tissues?

The β hydroxybutarate is converted into acetoacetate. This generates 1 NADH.
Succinyl-CoA gives up CoA to acetoacetate to form acetoacetyl-CoA.
Due to this, acetoacetyl-CoA bypasses the first 3 steps of β oxidation.
Finally thiolase cuts the acetoacetyl-CoA into 2 acetyl-CoA.
Ketones do not generate the same amount of ATP as normal β oxidation of a 4C fragment.

What is the difference between fasting, prolonged fasting and DM in terms of tissue usage?

Fasting - protein provides a lot of glucose for brain and RBC's. Adipose provides ketones and ffa's for heart, kidney and muscle.
Prolonged fasting - protein no longer provides glucose. Adipose provides ketones for brain and ffa's for muscle, heart and kidney. Ketone bodies end up in urine.
DM - Muscle breakdown is huge and brain uses glucose from this. Heart, kidney and muscle use ketones and ffa's. Ketones and glucose end up in urine.

How do you breakdown odd carbon unsaturated fatty acids? How do you change ATP yield calculation?

β oxidation is run until the cis double bond is at 3 position. Enoyl-CoA isomerase converts this into a trans double bond at 2 position which is now the normal acyl-CoA structure for β oxidation.
It yields 1.5 less ATP because FADH not generated in forming double bond.

How do you breakdown even carbon unsaturated fatty acids?

β oxidation is run until your trans double bond exist at 2 position and the existing cis double bond at 4 position. Using dienoyl-CoA reductase, you combine the 2 bonds into a 3 position trans bond and then using enoyl-CoA isomerase, you move the trans double bond to the 2 position. Now β oxidation can run as usual.

What happens if you have to degrade an odd numbered fatty acid? What enzymes and cofactors are used?

You use β oxidation until proprionyl-CoA exists. Using proprionyl-CoA carboxylase, ATP and biotin you add a carbon to form methylmalonyl-CoA. Then with methylmalonyl-CoA mutase and B12, you form succinyl-CoA.

What is the similarity between pyruvate carboxylase and proprionyl-CoA carboxylase?

Both use ATP and biotin.

How do you breakdown branched chain fatty acids?

Using alpha oxidation. The alpha carbon in this case is COOH and the β is CH2. You add OH to B carbon, you then convert it into C=O and then decarboxylate the compound to give CO2 and RCOOH.

What enzymes and cofactors are used in alpha oxidation?

Monooxygenase, O2, Fe2+ and ascorbate are used to form OH group on β carbon.
Then the OH is oxidized and NAD is reduced with a dehydrogenase enzyme.
Finally, ATP, NAD+ and ascorbate are used to decarboxylate the compound.

What genetic disease results when alpha oxidation is deficient?

Refsum's disease.

How do we form bile acids? What cofactors and enzymes are used?

We use ω oxidation to convert CH3 to COOH forming a dicarboxylic acid.
Monooxygenase and NADPH - notice NADPH

Where does fa synthesis occur? What about elongation of fatty acids and desaturation?

Synthesis occurs in the cytosol.
Elongation occurs in the mitochondria
Desaturation occurs in the ER.

What is the rate limiting step in fa synthesis? What enzyme and cofactors are required?

The formation of malonyl-CoA from acetyl-CoA.
Acetyl-CoA carboxylase, biotin, ATP and HCO3- are required for the formation.

Transfer the acetyl-CoA group on the FAS protein to malonyl-CoA on the ACP protein. Lose COOH to form β-ketoacyl.
Enzyme: β-ketoacyl synthetase.

How many CO2 are encorporated into a fatty acid?

None, the CO2 added in forming malonyl-CoA is lost when transferring acetyl-CoA to malonyl-CoA on the ACP protein.

What is the 2nd step of fa synthesis? What enzyme is required? What cofactor is required?

You convert the β carbon C=O to C-OH.
Enzyme: β-ketoacyl reductase
IMPORTANT: NADPH is used here for the first time.

What is the 3rd step of fa synthesis? What structure is formed? What type of double bond is formed?

Use a hydratase enzyme to take H2O off the C-OH and form a double bond btw alpha and β carbon.
Crotonyl-S-ACP
Trans double bond.

What is the 4th step of fa synthesis? What enzyme and cofactors are used?

Reduce the double bond btw α and β carbons to a single bond.
Enzyme: Enoyl reductase
NADPH is once again used right here

What 2 steps of fa synthesis do we use NADPH?

Reducing the β C=O to an OH (step 2)
Reducing the C=C to single bond (step 4).

What is the last step of fa synthesis?

Using a transferase, we move the acyl fragment from the ACP protein to the FAS protein so that a new malonyl-CoA can join at the ACP protein

How many acetyl-CoA, malonyl-CoA, NADPH, ATP and CO2 do we need to make palmitic acid?

8 acetyl-CoA
7 malonyl-CoA
14 NADPH (2 for each cycle, except for the first kind of)
7 ATP to form malonyl-CoA
7 CO2 to add to malonyl-CoA but none of these are encorporated, just used.

What is the source of NADPH for the following tissues?
Liver, brain, muscle

Liver gets it from the pentose phosphate shunt.
Brain gets it from cytosolic isocitrate dehydrogenase
Muscle gets it from malic enzyme, converting OAA into malate.

What is C75? How does it work?

It is a new drug for obesity. It blocks FAS which increases malonyl-CoA which inhibits certain neuropeptides responsible for appetite.

What are the steps of fatty acid elongation?

Reverse of β oxidation.
Thiolase to add acetyl-CoA to palmitic acid.
Dehydrogenase (EXCEPT WITH NADPH) to create OH group.
Hydratase to convert OH to a double bond.
Finally dehydrogenase (ONCE AGAIN WITH NADPH INSTEAD) to form a single bond.

How do you make unsaturated fatty acids? What enzyme is needed? What are the contraints?

Take glycerol, phosphorylate it with glycerokinase to form α-Glycerophosphate.
Add 2Acyl-CoA to form 1,2-Diacylglycerol (phosphatidic acid).
Use phosphatidate phosphohydrolase to remove the phosphate to form diglyceride.
Add acyl-CoA to form TGL (COMMON TO ALL PATHWAYS)

What method can all liver, adipose and intestines all use to make TGL's?

Take DHAP
Use α-Glycerophosphate dehydrogenase and NADH to convert C=O in middle to CH2-OH.
Now you have α-Glycerophosphate.
Convert this to phosphatidic acid and the rest is easy.

Why do alcoholics develop fatty livers?

Alcohol dehydrogenase generates NADH when converting ethanol to acetaldehyde.
Acetaldehyde is then converted into acetate by acetaldehyde dehydrogenase which also generates NADH.
Increases in NADH stimulate α-Glycerophosphate dehydrogenase which converts DHAP into α-Glycerophosphate. This increases TGL formation.

What enzyme breaks down TGL's in adipose tissue and what are the products formed?

Triglyceride lipase converts TGLs into diglycerides and ffa's.
Diglycerides are further broken down into glycerol and ffa's.

Why can't adipose tissue make TGL's out of glycerol?

Because it lacks glycerokinase which is required to convert glycerol into α-Glycerophosphate.
This is also why during fa breakdown in adipose tissue, the glycerol cannot be used and is sent to the liver for processing.

Where does the formation of glycerophospholipids occur?
What is a precursor to their formation?

In the ER.
1,2-diacylglycerol phosphate (phosphatidic acid)

When forming PC or PE, which compound is CDP activated?

The choline or ethanolamine is CDP activated and then added to the 1,2-diacylglycerol (diglyceride)

When forming PC or PE, do you use phosphatidic acid or diglyceride? What about PI?

You use diglyceride for PC and PE and you use phosphatidic acid for PI

When forming PI, which compound is CDP activated?

The phosphatidic acid is CDP activated in this case

How do you attach the CDP to ethanolamine or choline? What about CDP-phosphatidic acid?

The choline or ethanolamine is phosphorylated with ATP. Then CTP adds to the phosphorylated portion kicking off PPi and yielding CMP.
Well phosphatidic acid is already phosphorylated, so just add the CTP.

Which 2 phospholipids can you easily interconvert? How do you do this?

Phosphatidylethanolamine and phosphatidylserine.
To form PS, you just exchange the serine for ethanolamine.
To form PE from PS, you have to decarboxylate.

Name 2 ways to make phosphatidylcholine?

Add CDP-choline to diglyceride or use SAM to triple methylate PE.

What happens to the position of PE in the membrane as you methylate it?

PE moves to the outside, the more methyls you add to form PC.

Name 3 functions of phospholipids?

They form membranes for cellular structural support
PIP2 can be cleaved by phospholipase C to yield 2nd messengers IP3 and DAG.
You can cleave PI with phospholipase A2 to yield arachidonic acid for prostaglandin synthesis.

How does DAG cause it's 2nd messenger effects?

It activates protein kinase C which phosphorylates a number of protein products (including IRS proteins for insulin signalling)

How does IP3 cause it's 2nd messenger effects?

It binds to the ER and causes Ca2+ release which activates certain proteins and other protein kinases.

What is the precursor to all sphingolipids?
How do you make this?

Ceramide
Add serine to palmitic acid to form dehydrosphinganine. Then reduce with NADPH to form sphinganine and introduce a double bond for sphingosine.
Finally, the most important step, add a fatty acyl to the serine residue in an AMIDE BOND to form ceramide.

How do you make sphingomyelin? What about cerebrosides? What about sulfatide and gangliosides?

Add CDP choline to ceramide
Add UDP-gal or UDP-glu to ceramide
For sulfatide add the activated sulfur compound PAPS to cerebroside.
For gangliosides add a number of sugar residues and sialic acid.

What is wrong in Niemann-Pick's disease?

Sphingomyelinase is missing so sphingomyelin builds up

What is wrong in Gaucher's disease?

Lack of glucocerebrosidase
As a result glu-ceramide increases.

What is wrong in Tay-Sachs disease?

Lack of hexosaminidase A
Normally breaks down N-acetylgalactosamine ceramide to ceramide
N-acetyl builds up in cells.

Which organelles contain high levels of PC, sphingomyelin, cholesterol and cardiolipin

How do spleen macrophages detect aging RBC's?
How could you mimic the action?

RBC's normally have sialic acid attached to their surface proteins. This sialic acid has a negative charge and allows them to repel one another. When RBC's age, they lose this sialic acid as it breaks off. Macrophages can recognize this missing sialic acid and destroy the RBC.
If you use neuraminidase, you can remove the sialic acid enzymatically.

What is the very first step in cholesterol synthesis? What enzyme is used?

Take 2 acetyl-CoA and combine them into acetoacetyl-CoA in the reverse of the last step of B-oxidation.
The enzyme is thiolase which is actually named for the reverse reaction.

What is the second step of cholesterol synthesis? What enzyme is used?

The second step is adding another acetyl-CoA to acetoacetyl-CoA to form HMG-CoA.
The enzyme that does this is HMG-CoA synthase (same in ketone body formation)

What is the rate limiting step of cholesterol formation?
What enzyme is it?
What cofactors are used?
What control do we have of this enzyme?
Where does this reaction take place?

The rate limiting step is HMG-CoA reductase converting HMG-CoA into mevalonate.
HMG-CoA reductase uses NADPH and it controlled by cholesterol through negative feedback. In addition, this is where the statin drugs work.
This reaction takes place in the ER.

Discuss the formation of cholesterol from mevalonate including numbers?

Attach a fatty acid to the OH group on the A ring in the form of acyl-CoA.
Either ACAT (acylcholesterol acyl transferase) or LCAT (lecithin cholesterol acyl transferase).

Where are ACAT and LCAT found?

ACAT is found in the tissues and LCAT in the plasma.

Where does LCAT get it's substrate from?

LCAT cuts a monounsaturated fatty acid off of lecithin (PC) and transfers it to cholesterol.

What is the relationship with oleic acid and LCAT?

If you eat a diet rich in oleic acid, PC will have oleic acid at the R2 position.
LCAT will then take this oleic acid and transfer it to cholesterol and store the oleic acid. This is good.

What does a deficiency in LCAT cause?

It causes the serum free cholesterol to rise in the blood and the CholE to drop. Normally the ratio is 25% free, 75% CholE.

T/F ACAT uses lecithin to form cholesterol esters in the tissues?

False, ACAT can add any fatty acyl-CoA to cholesterol for storage.

What are the two major bile acids?
What is the main difference between them and cholesterol?

Cholic acid and chenodeoxycholic acid
The proprionic group of cholesterol was cleaved by ω and β oxidation. This left a COOH group at this position.
Other than that, numerous positions are hydroxylated.

What is the normal ratio of cholic acid to chenodeoxycholic acid?

80% cholic
20% chenodeoxycholic acid.

What are the steps of bile formation? Include rate limiting step and enzyme

Cholesterol is converted into 7-a-hydroxycholesterol by cholesterol 7-a-hydroxlase. THIS IS THE RATE LIMITING STEP.
The 7-a-hydroxycholesterol is then converted into 7-a-hydroxy-4-cholestene-3-one by a dehydrogenase, isomerase and NAD is needed. This step involves moving the double bond from the B to the A ring and oxidizing the OH group to C=O
This last compound is then converted into cholic acid and chenodeoxycholic acid.

What enzyme do we control when we control bile synthesis? What can we control it with?

7-a-hydroxylase which starts the conversion of cholesterol into bile acids.
Cholesterol is a positive stimulus for this enzyme and bile acids are a negative feedback.

How do we convert cholic acid to primary bile acids? Which primary bile acid is more common?
What happens if we shift this balance?

Using CoASH and ATP, we attach the CoA to the COOH group on cholic acid. Then with either glycine or taurine, we attach the aa to this same area kicking off the CoA. We now have primary bile acids like taurocholic acid or glycocholic acid.
(Note, you can do this with chenodeoxycholic acid too).
Glycocholic acid is 75% and Taurocholic acid is 25%.
You develop gall stones.

What time of bond exists between taurine/glycine and cholic acid? Why is this important?
What other compound has an amide bond between a fat and an aa?

An AMIDE bond
Bacteria can break this bond to help with excretion.
Ceramide - serine and palmitic acid or additional fatty acid added.

What apolipoprotein is specific to chylomicrons?
What is the disease called if you are missing it?

B-48
A betalipoproteinemia

What are endogenous and exogenous lipoproteins?

Endogenous - VLDL's
Exogenous - Chylomicrons

What enzyme is needed to convert a VLDL into an IDL?
What about converting IDL into LDL?

Lipoprotein lipase to remove TGL's from the VLDL
LCAT to transfer the cholesterol ester from HDL to IDL to form LDL. In the process cholesterol is given to the HDL.

What are the important apolipoproteins, where are they found and what is their function?

A-1 on HDLs and VLDLs. It 'A'ctivates LCAT
B-48 on chylomicrons. Needed for synthesis in intestines.
B-100 on LDLs, IDLs and VLDLs. 'B'inds to liver for remnant uptake.
C-1 is found on chylomicrons and VLDLs. Activates LCAT like A-1
C-11 is found on chylomicrons and VLDL's. Activation lipoprotein lipase.
E found on VLDLs, IDLs and chylomicrons. Also for liver recognition.

Discuss LDL receptor control in tissues

LDL binds to receptor.
Is taken up by cell by receptor mediated endocytosis
The cholesterol ester inside is broken down into free cholesterol and fatty acids.
This free cholesterol inhibits HMG-CoA reductase and also binds to ER to reduce LDL receptor synthesis.
The free cholesterol is then esterified with ACAT to be stored.

4 reasons why HDLs are good for you

1) Speed the formation of LDL's by cholesterol ester transfer with IDLs. This allows quicker storage of cholesterol.
2) They remove TGL's from chylomicrons quickly to leave the cholesterol rich remnant which is taken up by the liver.
3) They tranfer ApoCII and E to VLDLs and chylomicrons to cause faster activation of lipoprotein lipase and quicker uptake of remnants.
4) They take cholesterol from dead tissue and membranes back to the liver for excretion.

What is elevated in Type I hyperlipoproteinemia? What is the problem? Treatment?

It is a genetic deficiency in lipoprotein lipase so chylomicrons and serum TGL's are elevated. There is minor cholesterol elevation.
Reduce dietary fats

What is elevated in Type IIa hyperlipoproteinemia? What is the problem? Treatment?

IIa is an LDL receptor deficiency. Increased LDLs and cholesterol in the serum.
Statins, resins and diet.

What is elevated in Type IIb hyperlipoproteinemia? What is the problem? Treatment?

1) Formation of fatty streaks which is deposition in macrophages and smooth cells of the blood vessel.
2) Cholesterol esters, dermatin sulfate and platelets form calcified plaques which cause hardening of the arteries and clot formation.

What level of serum cholesterol predisposes you to an MI? What else does it predispose you to? What is the recommended level to stay below?

> 250mg/dl
Death from the 1st MI
200mg/dl

What are the primary factors increasing your risk for MI? What about secondary factors?

Cholesterol deposits in the tunica intima of the artery. This cholesterol gets oxidized and is taken up by macrophages, these are foam cells.
T-cells can also secrete cytokines which attract smooth muscle cells to ingest the oxidized LDL and form foam cells.

Why do alcohol and statin drugs help with reduced atherosclerosis?

Alcohol and statin drugs are both antioxidants that prevent oxidation of the B ring double bond of cholesterol.
In addition, statins block HMG-CoA reductase lowering the de novo synthesis of cholesterol.

What is the new marker for heart disease? Where does it come from? Does it correlate with serum cholesterol?

C-reactive protein (CRP) comes from the liver in response to vascular cells releasing interleukin-6 after damage.
It does not correlate well with serum cholesterol

It blocks COXI best but is also able to block COXII
Aspirin acetylates a serine residue in the active site of COXI. This is an irreversible chemical reaction.

What does tylenol block?

COXII

What enzymes do I use to make prostacyclin and thromboxane A2 from PGH2?

Prostacyclin synthase
Thromboxane synthase

Discuss how platelets aggregate? How does aspirin stop this?

Thrombin binds to the platelet and activates phospholipase A2.
This cuts arachidonic acid from PIP2 and COXI converts the arachidonic acid into PGG2 and then hydroperoxidase converts PGG2 into PGH2.
PGH2 is converted into thromboxane A2 by thromboxane synthase.
Thromboxane A2 causes platelets to become sticky.
Aspirin blocks the COXI step.

How does prostacyclin work? Where is it made and how?

PGI2 (prostacyclin) is made in the endothelium.
COXII converts arachidonic acid into PGG2 and then hydroperoxidase converts that into PGH2.
PGH2 is converted into PGI2 by prostacyclin synthase.
PGI2 binds to platelets and activates adenylate cyclase. This lowers cAMP which inhibits Phospholipase A2. No arachidonic acid, no thromboxane A2 preventing platelets from sticking.

Name 2 functions of thromboxane A2 and prostacyclin

TXA2 is a vasoconstrictor and platelet aggregator.
Prostacyclin is a vasodilator and causes platelets to repel.

How do I make leukotrienes?

Lipooxygenase converts arachidonic acid into either LTA4, LTB4 or LTC4. These are inflammatory mediators.
Lipooxygenase can also convert arachidonic acid into 5 or 10-HPETE - these are vasoconstrictors

What can I use to block leukotrienes?

Singulair blocks the action of leukotrienes.

What is saponification?

Using alkaline hydrolysis to break fatty esters into soap.
If the fat doesn't have any esters then it is non-saponifiable

What is the difference btw cis and trans fatty acids?

Cis lower the melting point because it creates a kink in the fatty acid.

What is the function of ∆3 cis-∆2 trans enoyl-CoA isomerase?

It is needed to break down unsaturated fatty acids because B-oxidation requires a trans double bond at the 2 position. The enzyme converts the cis double bond at the 3 position to the 2' trans double bond.

What does a deficiency in LCAT cause?

Low levels of serum cholesterol esters and lysolecithin.
High levels of serum cholesterol and lecithin.

How does a-ketoglutarate dehydrogenase differ from pyruvate dehydrogenase?

Pyruvate dehydrogenase has protein phosphatase activity

Why is oxygen still used when amytal is applied to mitochondria?

Amytal blocks complex I but complex II is still able to feed protons into Ubiquinone

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